SUMMARY Human fertility depends on the sequential differentiation of germline cells beginning with the formation of progenitors called human primordial germ cells (hPGCs), and ending with gametogenesis to create eggs and sperm. If hPGCs either do not develop correctly, or alternatively do not form at all, then problems with fertility will be guaranteed. Give the necessary role for hPGCs in germline cell development, understanding the fundamental basis of hPGC biology is essential to understanding fertility and treating infertility. Human PGCs develop during prenatal life. Therefore, the use of consented human fetal tissue is required to study the fundamental biology of hPGCs. In order to reduce the need for human fetal tissue in research, alternate models are required to reliably and reproducibly generate hPGCs in vitro. In this proposal, we aim to use human pluripotent stem cells (hPSCs) to model hPGC development. The differentiation of hPSCs into germline cells creates in vitro cell types called hPGC-like cells (hPGCLCs). This nomenclature is an acknowledgement to the origin of hPGCLCs as not being from human fetal tissue. The hPSC model and generation of hPGCLCs is currently used to decipher the cell and molecular events in hPGC specification. However, this model does not reliably recapitulate the next step in hPGC development, which is the global erasure of DNA methylation. Global erasure of DNA methylation in hPGCs is necessary for erasing epigenetic marks at imprinted genes, and in females regulating the timing of entrance into meiosis. Therefore, in order for the hPSC model to accurately and reliably model hPGC development, new approaches are required to promote complete global erasure of DNA methylation equivalent to what has been observed for hPGCs in vivo. To achieve this, our approach involves new methods for differentiating hPGCLCs, and comparing the methylome and transcriptome of hPGCLCs to pre-existing data sets of hPGCs. Once the technology for promoting hPGCLCs to complete DNA methylation erasure has been verified, the in vitro model will be poised for further study into human meiosis and gametogenesis, which collectively will serve to further reduce the need for fetal tissue use in research.